To study prevention of AIDS, we have a long-standing collaboration with George Pavlakis (Human Retrovirus Section, Vaccine Branch), and we have generated efficient DNA expression vectors, which are currently evaluated as vaccines against SIV and HIV. This work is based on the previous recognition that RNA elements (called INS) present within the gag/pol and env coding regions of HIV are responsible for nuclear retention and instability of the transcripts in the absence of Rev, and that these elements can be eliminated by changing the RNA composition without affecting the amino acid sequence. These RNA optimized gag and env expression vectors mediate the development of protective immune responses in vaccinated macaques when used as DNA only as well as DNA prime vaccine modality. We have now shown that that a combination of DNA vaccine vectors producing native and modified antigens are able to induce immune responses able to protect from high viremia in the rhesus macaque/SIVmac251 model. Current studies examine the role of these DNA vaccines as therapeutic modalities. In collaboration with other investigators we have studied immunogenicity of additional HIV and SIV genes either as DNAs or as part of viral vectors. We have shown that these DNAs provide an excellent prime in DNA prime-recombinant Herpes boost studies inducing protective immune responses. We have also shown that combinations of SIV DNA vaccine together with IL-12 or IL-15 as DNA adjuvants improve immunogenicity.Our goal is to dissect correlates of protective immunity, which will allow design of better vaccine strategies. Thus infected macaques, able to control viremia serve as model to study the underlying mechanisms. Among the 'controllers', animals infected with live-attenuated SIV strains provide an important model to study cellular and viral determinants that contribute to disease development and they also provide a unique tool to study mechanisms leading to protective immunity. We study the regulated expression of HIV, which is mediated via the viral Rev protein with the goal to evaluate the role of Rev in pathogenesis. Our discovery of other RNA export mechanisms (mediate via cellular RNA export factors and retrovial RNA export elements CTE, RTE as studied in project 1) provided the key tools to generate HIV and SIV variants that have the Rev/RRE regulatory system replaced. CTE or RTE provide us also with useful tools to achieve efficient expression of HIV and SIV structural protein in simple DNA vectors used as vaccine approaches against AIDS. Importantly, we found that our Rev-independent SIV strains are not pathogenic in rhesus macaques, which demonstrates that the Rev regulation plays an important role for SIV pathogenicity. In a recent long-term follow-up study we showed that such animals show persistent humoral and cellular SIV-specific immune responses, consistent with chronic infection but lack of pathogenicity. Multicolor flow cytometric analysis demonstrated preservation of the Central Memory subset of T-cells in the attenuated SIV-infected animals. This study demonstrates a potent, long-lasting control of the Rev-independent attenuated SIV in macaques. Replacement of the natural regulatory controls of Rev-RRE by the CTE provides a novel approach to lower the virulence of a pathogenic lentivirus. These studies provide critical information about the establishment and maintenance of host immune responses during chronic retroviral infections with distinct pathogenic outcomes. Macaques immunized with attenuated viruses develop persistent humoral and cellular immune responses, able to protect the animals from highly pathogenic SIV challenge. We are in the process to study the underlying mechanism. Therefore, these live-attenuated SIV strains provide us not only with a unique tool to dissect cellular and viral determinants that contribute to AIDS development, but are also useful for studying correlates of protective immunity.